Chromatography is a technique whereby a substance dissolved in a mobile phase is passed through a stationary phase to separate the substance so that it may be purified or identified, often from a complex mixture of substances. A substance includes, but is not limited to: (1) gases, (2) chemicals, (3) biological materials such as DNAs, carbohydrates, lipids, peptides, proteins or combinations thereof, (4) molecules, or (5) any combination of the aforementioned substances. A substance separated using chromatography may be analyzed by various means of detection which may be attached to the apparatus. Many variations in both the type of chromatography and the mechanism of separation presently exist and are actively used in research.
Countercurrent chromatography is based on liquid-liquid partitioning where both the mobile and stationary phases are in a liquid state. The technique separates compounds in a moving liquid phase from a stationary liquid phase held in coiled tubing and exposed to forces arising by rotation, for example, in a planetary centrifuge. Countercurrent chromatography has existed since the early 1970's and has been successfully employed to separate chemical compounds or small molecule substances. U.S. Pat. Nos. 3,784,467, 3,775,309, 6,503,398, 5,770,083, 5,354,473, 5,332,504 and 5,215,664. Sample substances are separated according to the solubility of the substances between the phases. Solvents may be mixed in varying volume ratios to form two stable immiscible phases that comprise the solvent system for the chromatography. Countercurrent chromatography provides a versatile method of separating or purifying small molecule substances; indeed, the prior art has been largely limited to small molecule separation, identification, or purification. While a few prior disclosures relate to countercurrent chromatography centrifuges that are alleged to separate large molecule substances, those lacked the ability to do so as efficiently and well as the instant invention. U.S. Pat. Nos. 4,714,554 and 5,104,531.
The earliest embodiment of countercurrent chromatography involved a series of columns fixed vertically and connected to one another by transfer tubing with a pump at one end. U.S. Pat. No. 3,784,467. The sample substance was dissolved in a mixture of the solid and stationary phases in another column that was then connected to the pump by transfer tubing and placed before the other columns in the series.
A subsequent invention showed how to configure continuous tubing for solvent entry and exit, without a rotating seal, in helical coils that rotate around a central axis. U.S. Pat. No. 3,775,309. In one revolution, the apparatus accomplished a twisting and untwisting action in a planetary motion that mixed the phases. During a single revolution in that system, inside a tubing coil filled with a dual phase solvent, the lighter phase migrates to one end (head) and the heavier phase migrates to the other end (tail) depending on (1) the rotational direction of the coil (clockwise or counterclockwise) and (2) the rotational direction of the rotor (clockwise or counterclockwise). The mobile phase is pumped into the end against the direction in which the stationary phase prefers to move; thus, the stationary phase at equilibrium is retained at 40-80% of the total volume of the coil. Excess of the mobile phase elutes out the other end. Current embodiments or applications of planetary CCC centrifuges involve different orientations of the helical tubing. U.S. Pat. Nos. 3,994,805, 4,430,216, 4,532,039 and 4,714,554.
In recent years, the helical tubing or spool has been replaced by spirally-organized tubing held in place by a frame called a spiral tubing support. U.S. Pat. Pub. No. US 2005/0242040. Presently, construction of the spiral tubing support is slow and laborious. The first type of that apparatus was built by crafting spiral channels ground two inches deep into light aluminum and radial channels to hold Teflon® tubing wound in four spirals in one plane. The advantages of the spiral tubing support are (1) lack of leakage through sandwiched plates with flow channels and (2) higher resolution of substances due to the smaller diameter of the tubing.